Abstract
The organoarsenical feed additive 4-hydroxy-3-nitrobenzenearsonic acid (roxarsone [ROX]) is widely used and released into the environment. We previously showed a two-step pathway of ROX transformation by Enterobacter sp. strain CZ-1 involving the reduction of ROX to 3-amino-4-hydroxyphenylarsonic acid (3-AHPAA) and the acetylation of 3-AHPAA to N-acetyl-4-hydroxy-m-arsanilic acid (N-AHPAA) (K. Huang, H. Peng, F. Gao, Q. Liu, et al., Environ Pollut 247:482-487, 2019, https://doi.org/10.1016/j.envpol.2019.01.076). In this study, we identified two nhoA genes (nhoA1 and nhoA2), encoding N-hydroxyarylamine O-acetyltransferases, as responsible for 3-AHPAA acetylation in Enterobacter sp. strain CZ-1. The results of genetic disruption and complementation showed that both nhoA genes are involved in ROX biotransformation and that nhoA1 is the major 3-AHPAA acetyltransferase gene. Quantitative reverse transcription-PCR analysis showed that the relative expression level of nhoA1 was 3-fold higher than that of nhoA2 Each of the recombinant NhoAs was overexpressed in Escherichia coli BL21 and homogenously purified as a dimer by affinity chromatography. Both purified NhoAs catalyzed acetyl coenzyme A-dependent 3-AHPAA acetylation. The Km values of 3-AHPAA for NhoA1 and NhoA2 were 151.5 and 428.3 μM, respectively. Site-directed mutagenesis experiments indicated that two conserved arginine and cysteine residues of each NhoA were necessary for their enzyme activities.IMPORTANCE Roxarsone (ROX) is an organoarsenic feed additive that has been widely used in poultry industries for growth promotion, coccidiosis control, and meat pigmentation improvement for more than 70 years. Most ROX is excreted in the litter and dispersed into the environment, where it is transformed by microbes into different arsenic-containing compounds. A major product of ROX transformation is N-acetyl-4-hydroxy-m-arsanilic acid (N-AHPAA), which is also used as a clinical drug for treating refractory bacterial vaginosis. Here, we report the cloning and functional characterization of two genes encoding N-hydroxyarylamine O-acetyltransferases, NhoA1 and NhoA2, in Enterobacter sp. strain CZ-1, which catalyze the acetylation of 3-amino-4-hydroxyphenylarsonic acid (3-AHPAA) formed by the reduction of ROX to N-AHPAA. This study provides new insights into the function of N-hydroxyarylamine O-acetyltransferase in the transformation of an important organoarsenic compound.